Amino Acids From Biological Samples

Studies by Heathcote (1979) indicate that quantitative densitometric TLC analyses of amino acids will provide results equivalent to that obtained from automated amino acid analysis procedures. In a study by Mack et al. (1979) on amino acids in mosquitoes, qualitative TLC procedures were first used to determine amino acid profiles in the tissue and hemolymph, and, subsequently, the automated amino acid analyzer was applied for quantification. The study of Mack et al. (1979) should be useful to those interested in qualitative and quantitative analysis of amino acids from biological samples. 

Source From Optimization of dansyl derivatization and chromatographic conditions in the determination of neuroactive amino acids of biological samples, in Clin. Chim. Acta. ... 

Amines other than a-amino acids will also give a colour reaction with ninhydrin but without the production of carbon dioxide. Thus /3-, y-, 8- and e-amino acids and peptides react more slowly than a-amino acids, to give the blue complex, while imino acids result in the formation of a yellow-coloured product which can be measured at 440 nm. Removal of substances such as protein, ammonia and urea from biological samples may be necessary in quantitative work because they also react in a similar manner. 

There is a way of bringing amino acids from the biological matrix into an aqueous/organic surrounding - by deproteinization with acetonitrile or alcohol. This is the preferred method of preparing samples for MS/MS. 

The reaction of amines and amino acids with orthophthaldehyde has been widely used in postcolumn and precolumn derivatization in analyses of foods (99-104) and in analyses of peptides from biological samples. Figure 2 (87) presents a chromatogram for OPA derivatives of tryptic peptides from two proteins. The sensitivity of the method was on the order of picomoles. The authors have themselves performed postcolumn OPA derivatization of low-molecular-weight peptides from blue cheeses separated by reversed-phase chromatography (86). 

DHA can be separated from AA and most ionic compounds by ion exchange columns. Such columns do not separate DHA from other AA metabolites and neutral carbohydrates. DHA can be separated from these compounds by reverse phase HPLC using water or water-acetonitrile eluants. Good separations of DHA from biological samples can be expected to be achieved by HPLC. Detection is a problem since UV absorption is inadequate. The red chromophore with amino acids is not very sensitive. Perhaps DHA could be reduced to AA after separation and detected by the strong 263-nm absorption of AA or by an electrochemical detector. DHA levels and DHA/AA ratios are probably quite important in biology and medicine, and good procedures for these assays are of considerable interest. 

The determination of amino acids in various samples is a usual task in many research, industrial, quality control, and service laboratories. Hence, there is a substantial interest in the HPLC analysis of amino acids from many diverse areas like biochemistry, biotechnology, food quahty control, diagnostic services, neuro-chemistry/biology, and so forth. As a result, the separation of amino acids is probably the most extensively studied and best developed chromatographic separation in biological sciences. The most known system is the separation on a cation-exchange column and... 

Bhushan (1991) on amino acids and their derivatives their new review includes references through 1994. Jain (1996) has reviewed studies on the applications of TLC to amino acid analysis of biological fluids and tissues. Such analyses are important in making diagnoses of inborn errors of amino acid metabolism. It was noted by Jain (1996) that TLC has proved useful to screen and quantify abnormal amounts of free amino acids in blood and urine samples. Shalaby (1996) in his chapter on TLC in food analysis has provided some protocols on amino acid analysis following the hydrolysis of protein samples. Fried and Haseeb (1996) in their chapter on TLC in parasitology have provided some information on the analysis of free pool amino acids from tissues of parasites and from the hemo-lymph of mosquitoes infected with Plasmodium (the malaria organism). 

Lipid extracts obtained from biological samples, as aforementioned, tend to contain significant amounts of nonlipid contaminants, such as sugars, amino acids, urea, and salts. These must be removed before the lipids are analyzed. A common and classical approach is to use a simple washing procedure devised by Folch, Lees and Sloane Stanley [17], in which a chloroform-methanol (2 1, v/v) extract is shaken and equilibrated with one-fourth its volume of saline solution (i.e., 0.88% potassium chloride in water). The mixture partitions into two layers, of which the lower phase is comprised of chloroform-methanol-water in a proportion of 86 14 1 (by volume) and contains virtually all of the lipids, while the upper phase consists of the same solvents in the proportion of 3 48 47 (by volume), respectively, and contains much of the nonlipid contaminants. It is important that the proportion of chloroform, methanol, and water in the combined phases should be as close as possible to 8 4 3 (by volume), otherwise selective losses of lipids may occur. If a second wash of the lower phase is needed to remove any remaining contaminants, a mixture of similar composition to that of the upper phase should be used, i.e., methanol-saline solution (1 1, v/v). 

In addition to MALDI-TOF and LC-MS/MS, SELDI-TOF-MS can also be used to determine expression profiling of various biological samples, such as serum or plasma for early detection of infection. Serum proteomic profiling assay, for instance, has been used to distinguish patients with acute SARS (severe acute respiratory syndrome) from patients with fever and influenza with 100% accuracy [16]. A major limitation of SELDI-TOF-MS, however, is that it cannot be used for direct amino acid sequence identification of the biomarker proteins, necessitating further sample fractionation and protein purification. 

---